Ethanol is known to alter the biochemical and biophysical properties of cellular membranes. In this investigation the mechanisms of ethanol-induced membrane potential changes in brain synaptosomes and rat thymocytes were studied with fluorescence spectrophotometry using membrane potential-sensitive dyes. The effect of ethanol on membrane potential in synaptosomes was studied using rhodamine 6G fluorescence. The fluorescence signal responds to the depolarization produced by increasing concentration of K+ outside the synaptosomes. The fluorescence intensity increases with increasing [K+]O from 5mM to 60mM and then levels off at higher concentrations. When the [Na+]O concentration was changed from 0 to 137 mM, keeping [K+]O constant at 5 mM, no change is observed in fluorescence. On addition of ethanol, the fluorescence intensity increased. There was a significant change (15%) in fluorescence intensity with as low as 10 mM ethanol. The fluorescence intensity increase as ethanol concentration was varied from 10 to 100 mM. Ethanol had no effect on depolarized (80 mM KCl) synaptosomes. The effect of ethanol on membrane depolarization was similar to K+ depolarization. In the absence of synaptosomes ethanol had no effect in the fluorescence intensity of dye. In lysed or 12 hr. old synaptosomes, there was no change in fluorescence either with [K+]O or ethanol. We concluded that (1) Rhodamine 6G fluorescence can be used to monitor the change in membrane potential in synaptosomes (2) low doses of ethanol produces changes in membrane potential in synaptosomes isolated from rat brain.